Electrochemical sensor using intercalative, redox-active moieties

ABSTRACT

Compositions and methods for electrochemical detection and localization of genetic point mutations and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.

2. RELATED APPLICATION

[0001] This application is a continuation-in part of Ser. No.60/043,146, filed Apr. 9, 1997.

1. GOVERNMENT RIGHTS

[0002] The U.S. Government has certain rights in this invention pursuantto Grant No. GM 49216 awarded by the National Institute of Health.

3. FIELD OF THE INVENTION

[0003] The present invention relates to the detection and localizationof base-pair mismatches and other perturbations in base-stacking withinan oligonucleotide duplex.

4. DESCRIPTION OF RELATED ART

[0004] It is now well known that mutations in DNA can lead to severeconsequences in metabolic functions (e.g., regulation of geneexpression, modulation of protein production) which ultimately areexpressed in a variety of diseases. For example, a significant number ofhuman cancers are characterized by a single base mutation in one of thethree ras genes (Bos, 1989). In order to unravel the genetic componentsof such diseases, it is of utmost importance to develop DNA sensors thatare capable of detecting single-base mismatches rapidly and efficientlyand to establish routine screening of disease-related genetic mutationsbased on such sensors (Skogerboe, 1993; Southern, 1996; Chee, 1996; Eng,1997).

[0005] Various methods that have been developed for the detection ofdifferences between DNA sequences rely on hybridization events todifferentiate native versus mutated sequences and are limited by thesmall differences in base-pairing energies caused by point mutationswithin extended polynucleotides (Millan, 1993; Hashimoto, 1994; Xu,1995; Wang, 1996; Lockhart, 1996; Alivisatos, 1996; Korriyoussoufi,1997; Elghanian, 1997; Lin, 1997; Herne, 1997). Typically, a nucleicacid hybridization assay to determine the presence of a particularnucleotide sequence (i.e. the “target sequence”) in either RNA or DNAcomprises a multitude of steps. First, an oligonucleotide probe having anucleotide sequence complementary to at least a portion of the targetsequence is labeled with a readily detectable atom or group. When thelabeled probe is exposed to a test sample suspected of containing thetarget nucleotide sequence, under hybridizing conditions, the targetwill hybridize with the probe. The presence of the target sequence inthe sample can be determined qualitatively or quantitatively in avariety of ways, usually by separating the hybridized and non-hybridizedprobe, and then determining the amount of labeled probe which ishybridized, either by determining the presence of label in probe hybridsor by determining the quantity of label in the non-hybridized probes.Suitable labels may provide signals detectable by luminescence,radioactivity, colorimetry, x-ray diffraction or absorption, magnetismor enzymatic activity, and may include, for example, fluorophores,chromophores, radioactive isotopes, enzymes, and ligands having specificbinding partners. However, the specific labeling method chosen dependson a multitude of factors, such as ease of attachment of the label, itssensitivity and stability over time, rapid and easy detection andquantification, as well as cost and safety issues. Thus, despite theabundance of labeling techniques, the usefulness, versatility anddiagnostic value of a particular system for detecting a material ofinterest is often limited.

[0006] Some of the currently used methods of mismatch detection includesingle-strand conformation polymorphism (SSCP) (Thigpen, 1992; Orita,1989), denaturing gradient gel electrophoresis (DGGE) (Finke, 1996;Wartell, 1990; Sheffield, 1989), RNase protection assays (Peltonen andPulkkinen, 1986; Osborne, 1991), allele-specific oligonucleotides (Wu,1989), allele-specific PCR (Finke, 1996), and the use of proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991).

[0007] In the first three methods, the appearance of a newelectrophoretic band is observed by polyacrylamide gel electrophoresis.SSCP detects the differences in speed of migration of single-strandedDNA sequences in polacrylamide gel electrophoresis under differentconditions such as changes in pH, temperature, etc. A variation in thenucleotide base sequence of single-stranded DNA segments (due tomutation or polymorphism) may lead to a difference in spatialarrangement and thus in mobility. DGGE exploits differences in thestability of DNA segments in the presence or absence of a mutation.Introduction of a mutation into double-stranded sequences creates amismatch at the mutated site that destabilizes the DNA duplex. Using agel with an increasing gradient of formamide (denaturation gradientgel), the mutant and wild-type DNA can be differentiated by theiraltered migration distances. The basis for the RNase protection assay isthat the RNase A enzyme cleaves mRNA that is not fully hybridized withits complementary strand, whereas a completely hybridized duplex isprotected from RNase A digestion. The presence of a mismatch results inincomplete hybridization and thus cleavage by RNase A at the mutationsite. Formation of these smaller fragments upon cleavage can be detectedby polyacrylamide gel electrophoresis. Techniques based on mismatchdetection are generally being used to detect point mutations in a geneor its mRNA product. While these techniques are less sensitive thansequencing, they are simpler to perform on a large number of tumorsamples. In addition to the RNase A protection assay, there are otherDNA probes that can be used to detect mismatches, through enzymatic orchemical cleavage. See, e.g., Smooker and Cotton, 1993; Cotton, 1988;Shenk, 1975. Other enzymatic methods include for example the use of DNAligase which covalently joins two adjacent oligonucleotides which arehybridized on a complementary target nucleic acid, see, for exampleLandegren (1988). The mismatch must occur at the site of ligation.

[0008] Alternatively, mismatches can also be detected by shifts in theelectrophoretic mobility of mismatched duplexes relative to matchedduplexes (Cariello, 1988). With either riboprobes or DNA probes, thecellular mRNA or DNA which may contain a mutation can be amplified usingpolymerase chain reaction (PCR) prior to hybridization. Changes in DNAof the gene itself can also be detected using Southern hybridization,especially if the changes are gross rearrangements, such as deletionsand insertions.

[0009] DNA sequences of the specified gene which have been amplified byuse of PCR may also be screened using allele-specific oligonucleotideprobes. These probes are nucleic acid oligomers, each of which iscomplementary to a corresponding segment of the investigated gene andmay or may not contain a known mutation. The assay is performed bydetecting the presence or absence of a hybridization signal for thespecific sequence. In the case of allele-specific PCR, the PCR techniqueuses unique primers which selectively hybridize at their 3′-ends to aparticular mutated sequence. If the particular mutation is not present,no amplification product is observed.

[0010] In addition, restriction fragment length polymorphism (RFLP)probes for the gene or surrounding marker genes can be used to scorealteration of an allele or an insertion in a polymorphic fragment.However, since the recognition site of restriction endonucleases rangesin general between 4 to 10 base pairs, only a small portion of thegenome is monitored by any one enzyme.

[0011] Another means for identifying base substitution is directsequencing of a nucleic acid fragment. The traditional methods are basedon preparing a mixture of randomly-terminated, differentially labeledDNA fragments by degradation at specific nucleotides, or by dideoxychain termination of replicating strands (Maxam & Gilbert, 1980; Sanger,1977). Resulting DNA fragments in the range of 1 to 500 basepairs arethen separated on a gel to produce a ladder of bands wherein theadjacent samples differ in length by one nucleotide. The other methodfor sequencing nucleic acids is sequencing by hybridization (SBH,Drmanac, 1993). Using mismatch discriminative hybridization of shortn-nucleotide oligomers (n-mers), lists of constitutent n-mers may bedetermined for target DNA. The DNA sequence for the target DNA may beassembled by uniquely overlapping scored oligonucleotides. Yet anotherapproach relies on hybridization to high-density arrays ofoligonucleotides to determine genetic variation. Using a two-colorlabeling scheme simultaneous comparison of a polymorphic target to areference DNA or RNA can be achieved (Lipshutz, 1995; Chee, 1996; Hacia,1996).

[0012] Each of these known prior art methods for detecting base pairmismatches has limitations that affect adequate sensitivity, specificityand ease of automation of the assay. In particular, these methods areunable to detect mismatches independent of sequence composition andrequire carefully controlled conditions, and most methods detectmultiple mismatches only. Additional shortcomings that limit thesemethods include high background signal, poor enzyme specificity, and/orcontamination.

[0013] Over the last decade, attention has also focused on DNA as amedium of charge transfer in photoinduced electron transfer reactionsand its role in mutagenesis and carcinogenesis. For example, studieswere performed using various octahedral metal complexes (which bindtightly to DNA by intercalation) as donors and acceptors forphotoinduced electron transfer. Dppz complexes of ruthenium, osmium,cobalt, nickel, and rhenium showed tight intercalative binding andunique photophysical and electrochemical properties. No photoluminesencewas observed upon irradiation of the metal complexes in aqueous solutionin absence of DNA (as a result of quenching by proton transfer from thesolvent), whereas in the presence of DNA excitation of the complexafforded significant, long-wavelength emission (because now theintercalated complex was protected from quenching). Studies usingrhodium intercalators containing phenanthrenequinone-diimine (phi)ligands displayed tight DNA binding by preferential intercalation, somewith affinities and specifities approaching DNA-binding proteins.

[0014] Photoinduced electron transfer using DNA as a molecular bridgehas been established in various systems. Using metal complexesintercalated into the base stack of DNA as donor and acceptor it hasbeen proposed that the DNA π-stack could promote electron transfer atlong range. Additionally, the products of redox-triggered reactions ofDNA bases have been detected at sites remote from intercalating oxidants(Hall, 1996; Dandliker, 1997; Hall, 1997; Arkin, 1997). For example, ithas been shown that a metallointercalator can promote oxidative DNAdamage through long-range hole migration from a remote site. OligomericDNA duplexes were prepared with a rhodium intercalator covalentlyattached to one end and separated spatially from 5′-GG-3′ doublet sitesof oxidation. Rhodium-induced photooxidation occurred specifically atthe 5′-G in the 5′-GG-3′ doublets and was observed up to 37 Å away fromthe site of rhodium intercalation. In addition it was found that rhodiumintercalators excited with 400 nm light, initiated the repair of athymine dimer incorporated site-specifically in the center of asynthetic 16-mer oligonucleotide duplex. The repair mechanism wasthought to proceed via oxidation of the dimer by the intraligand excitedstate of the rhodium complex, in which an electron deficiency (hole) islocalized on the intercalated phi ligand. Like electron transfer betweenmetallointercalators, the efficiencies of long-range oxidative processeswere found to be remarkably sensitive to the coupling of the reactantsinto the base stack (Holmlin, 1997) and depended upon the integrity ofthe base stack itself (Kelley, 1997c, 1997d; Hall, 1997; Arkin, 1997) aswell as on the oxidation potential. Perturbations caused by mismatchesor bulges greatly diminished the yields of DNA-mediated chargetransport.

[0015] Other studies have reported electron transfer through DNA usingnonintercalating ruthenium complexes coordinated directly toamino-modified sugars at the terminal position of oligonucleotides(Meade, 1995). In this system it was suggested that electron transfer isprotein-like. In proteins, where the energetic differences in couplingdepend upon σ-bonded interactions, small energetic differences betweensystems do not cause large differences in electronic coupling. In theDNA double helix however, π-stacking can contribute to electroniccoupling such that small energetic differences could lead to largedifferences in coupling efficiency. Most recently, Lewis and coworkersmeasured rates of photo-oxidation of a guanine base in a DNA hairpin byan associated stilbene bound at the top of the hairpin (Lewis, 1997). Bysystematically varying the position of the guanine base within thehairpin and measuring the rate of electron transfer, a value for β, theelectronic coupling parameter, could be made. Here, β was found to beintermediate between that seen in proteins, with σ bonded arrays, andthat found for a highly coupled π-bonded array.

[0016] Electrochemical studies of small molecule/DNA complexes havefocused primarily on solution-phase phenomena, in which DNA-inducedchanges in redox potentials and/or diffusion constants of organic andinorganic species have been analyzed to yield association constants(Carter, 1989, 1990; Rodriguez, 1990; Welch, 1995; Kelly, 1986;Molinier-Jumel, 1978; Berg, 1981; Plambeck, 1984). In addition, rates ofguanine oxidation catalyzed by electrochemically oxidizedtransition-metal complexes have been used to evaluate the solventaccessibility of bases for the detection of mismatches in solution(Johnston, 1995). Electrochemical signals triggered by the associationof small molecules with DNA have also been applied in the design ofother novel biosensors. Toward this end, oligonucleotides have beenimmobilized on electrode surfaces by a variety of linkages for use inhybridization assays. These include thiols on gold (Hashimoto, 1994a,1994b; Okahata, 1992), carbodiimide coupling of guanine residues onglassy carbon (Millan, 1993), and alkane bisphosphonate films onAl³⁺-treated gold (Xu, 1994, 1995). Both direct changes in mass(measured at a quartz crystal microbalance) (Okahata, 1992) and changesin current (Hashimoto, 1994a, 1994b; Millan, 1993) or electrogeneratedchemiluminesence (Xu, 1994, 1995) due to duplex-binding molecules havebeen used as reporters for double stranded DNA. Gold surfaces modifiedwith thiolated polynucleotides have also been used for the detection ofmetal ions and DNA-binding drugs (Maeda, 1992, 1994).

[0017] Other known electrochemical sensors used in an increasing numberof clinical, environmental, agricultural and biotechnologicalapplications include enzyme based biosensors. Amperometric enzymeelectrodes typically require some form of electrical communicationbetween the electrode and the active site of the redox enzyme that isreduced or oxidized by the substrate. In one type of enzyme electrode, anon-natural redox couple mediates electron transfer from thesubstrate-reduced enzyme to the electrode. In this scheme, the enzyme isreduced by its natural substrate at a given rate; the reduced enzyme isin turn, rapidly oxidized by a non-natural oxidizing component of aredox couple that diffuses into the enzyme, is reduced, diffuses out andeventually diffuses to an electrode where it is oxidized.

[0018] Electrons from a substrate-reduced enzyme will be transferredeither to the enzyme's natural re-oxidizer or, via the redox-centers ofthe polymer to the electrode. Only the latter process contributes to thecurrent. Thus, it is desirable to make the latter process fast relativeto the first. This can be accomplished by (a) increasing theconcentration of the redox centers, or (b) assuring that these centersare fast, i.e. that they are rapidly oxidized and reduced.

[0019] Most natural enzymes are not directly oxidized at electrodeswithout being destroyed, even if the latter are maintained at stronglyoxidizing potentials. Also they are not reduced at strongly reducingpotentials without being decomposed. It has, however, been shown thatenzymes can be chemically modified by binding to their proteins redoxcouples, whereupon, if in the reduced state, they transfer electrons toan electrode. It has also been shown that when redox polycations insolution electrostatically complex polyanionic enzymes, electrons willflow in these complexes from the substrate to the enzyme, and from theenzyme through the redox polymer, to an electrode. In addition, systemshave been developed where a redox-active polymer, such aspoly(vinyl-pyridine), has been introduced which electrically connectsthe enzyme to the electrode. In this case, the polycationic redoxpolymer forms electrostatic complexes with the polyanionic glucoseoxidase in a manner mimicking the natural attraction of some redoxproteins for enzymes, e.g., cytochrome c for cytochrome c oxidase.

[0020] The present invention provides a new approach for the detectionof mismatches based on charge transduction through DNA. Thiselectrochemical method is based on DNA-mediated electron transfer usingintercalative, redox-active species and detects differences inelectrical current or charge generated with fully base-paired duplexesversus duplexes containing a base-stacking perturbation, such as amismatch. Carried out at an addressable multielectrode array, thismethod allows the processing of multiple sequences in the course of asingle measurement, thus significantly improving the efficiency ofscreening for multiple genetic defects. Most importantly, the assayreports directly on the structural difference in base pair stackingwithin the hybridized duplex, rather than on a thermodynamic differencebased on the condition-dependent hybridization event itself.Consequently, mismatch detection becomes independent of the sequencecomposition and sensors based on this approach offer fundamentaladvantages in both scope and sensitivity over any other existingmethods.

5. SUMMARY OF THE INVENTION

[0021] The present invention provides a highly sensitive and accuratemethod for the detection of genetic point mutations in nucleic acidsequences and its application as a biosensor. In particular, theinvention relates to electrodes that are prepared by modifying theirsurfaces with oligonucleotide duplexes combined with an intercalative,redox-active species and their use as sensors based on anelectrochemical process in which electrons are transferred between theelectrode and the redox-active species.

[0022] One aspect of the invention relates to methods for determiningthe presence of point mutations sequentially in a series ofoligonucleotide duplexes using an intercalative, redox-active moiety. Apreferred method comprises the steps of: (a) contacting at least onestrand of a first nucleic acid molecule with a strand of a secondnucleic acid molecule under hybridizing conditions, wherein one of thenucleic acid molecules is derivatized with a functionalized linker, (b)depositing this duplex onto an electrode or an addressablemultielectrode array, (c) contacting the adsorbed duplex whichpotentially contains a base-pair mismatch with an intercalative,redox-active moiety under conditions suitable to allow complexformation, (d) measuring the amount of electrical current or chargegenerated as an indication of the presence of a base-pair mismatchwithin the adsorbed duplex, (e) treating the complex under denaturingconditions in order to separate the complex, yielding a monolayer ofsingle-stranded oligonucleotides, and (f) rehybridizing thesingle-stranded oligonucleotides with another target sequence. Steps (c)through (f) can then be repeated for a sequential analysis of variousoligonucleotide probes. Attenuated signals, as compared to the observedsignals for fully base-paired, i.e. wild-type, sequences, willcorrespond to mutated sequences.

[0023] In some instances, it may be desirable to crosslink theintercalative, redox-active species to the duplex and perform the assaycomprised of steps (a) through (d) only.

[0024] Another preferred method relates to the detection of pointmutations utilizing electrocatalytic principles. More specifically, thismethod utilizes an electrode-bound double-stranded DNA monolayer whichis immersed in a solution comprising an intercalative, redox-activespecies, which binds to the monolayer surface, and a non-intercalativeredox-active species which remains in solution. This method comprisesthe steps of: (a) contacting at least one strand of a first nucleic acidmolecule with a strand of a second nucleic acid molecule underhybridizing conditions, wherein one of the nucleic acid molecules isderivatized with a functionalized linker, (b) depositing this duplexwhich potentially contains a base-pair mismatch onto an electrode or anaddressable multielectrode array, (c) immersing this complex in anaqueous solution comprising an intercalative, redox-active moiety and anon-intercalative, redox-active moiety under conditions suitable toallow complex formation, (d) measuring the amount of electrical currentor charge generated as an indication of the presence of a base-pairmismatch within the adsorbed duplex, (e) treating the complex underdenaturing conditions in order to separate the complex, yielding amonolayer of single-stranded oligonucleotides, and (f) rehybridizing thesingle-stranded oligonucleotides with another target sequence. Steps (c)through (f) can then be repeated for a sequential analysis of variousoligonucleotide probes. Utilizing this method, pronounced currents andthus increased signals will be observed due to the electrocatalyticreduction of the non-intercalative, redox-active moiety by thesurface-bound, redox-active moiety.

[0025] Yet another aspect of the invention relates to a method ofdetecting the presence or absence of a protein and comprises the stepsof: (a) contacting at least one strand of a first nucleic acid moleculewith a strand of a second nucleic acid molecule under hybridizingconditions, wherein one of the nucleic acid molecules is derivatizedwith a functionalized linker and wherein the formed duplex is designedsuch to contain the recognition site of a nucleic acid-binding proteinof choice, (b) depositing this duplex onto an electrode or anaddressable multielectrode array, (c) contacting the adsorbed duplexwith an intercalative, redox-active moiety under conditions suitable toallow complex formation, (d) potentially crosslinking the intercalative,redox-active moiety to the duplex, (e) immersing the complex in a firstsample solution to be analyzed for the presence of the nucleicacid-binding protein, (f) measuring the amount of electrical current orcharge generated as an indication of the presence or absence of thenucleic acid-binding protein in the sample solution, (g) treating thecomplex under appropriate conditions to remove the nucleic acid-bindingprotein, and (h) immersing it in a second sample solution to be analyzedfor the presence of the nucleic acid-binding protein in order toseparate the complex. Steps (e) through (h) can then be repeated for asequential analysis of various sample solutions. Attenuated signals, ascompared to signals measured for a reference solution without thenucleic acid-binding protein, indicate the presence of the nucleicacid-binding protein which is binding to its recognition site, thuscausing a perturbation in base-stacking.

[0026] The invention also relates to the nature of the redox-activemoieties. The requirements of a suitable intercalative, redox-activemoiety include the position of its redox potential with respect to thewindow within which the oligonucleotide-surface linkage is stable, aswell as the synthetic feasibility of covalent attachment to theoligonucleotide. In addition, chemical and physical characteristics ofthe redox-active intercalator may promote its intercalation in asite-specific or a non-specific manner. In a preferred embodiment, theredox-active species is in itself an intercalator or a larger entity,such as a nucleic acid-binding protein, that contains an intercalativemoiety.

[0027] The nature of the non-intercalative, redox-active species for theelectrocatalysis based assays depends primarily on the redox potentialof the intercalative, redox-active species utilized in that assay.

[0028] Yet another aspect of the invention relates to the compositionand length of the oligonucleotide probe and methods of generating them.In a preferred embodiment, the probe is comprised of two nucleic acidstrands of equal length. In another preferred embodiment the two nucleicacid strands are of uneven length, generating a single-stranded overhangof desired sequence composition (i.e. a “sticky end”). The length of theoligonucleotide probes range preferably from 12 to 25 nucleotides, whilethe single-stranded overhangs are approximately 5 to 10 nucleotides inlength. These single-stranded overhangs can be used to promotesite-specific adsorption of other oligonucleotides with thecomplementary overhang or of enzymes with the matching recognition site.

[0029] The invention further relates to methods of creating a spatiallyaddressable array of adsorbed duplexes. A preferred method comprises thesteps of (a) generating duplexes of variable sequence composition thatare derivatized with a functionalized linker, (b) depositing theseduplexes on different sites on the multielectrode array, (c) treatingthe complex under denaturing conditions to yield a monolayer ofsingle-stranded oligonucleotides, and (d) hybridizing thesesingle-stranded oligonucleotides with a complementary target sequence.Another preferred method comprises the steps of (a) depositing 5 to 10base-pair long oligonucleotide duplexes that are derivatized on one endwith a functionalized linker and contain single-stranded overhangs(approximately 5 to 10 nucleotides long) of known sequence compositionat the opposite end onto a multielectrode array, and (b) contactingthese electrode-bound duplexes under hybridizing conditions withsingle-stranded or double-stranded oligonucleotides that contain thecomplementary overhang.

[0030] Another aspect of the invention is directed towards the nature ofthe electrode, methods of depositing an oligonucleotide duplex (with orwithout a redox-active moiety adsorbed to it) onto an electrode, and thenature of the linkage connecting the oligonucleotide duplex to theelectrode. In a preferred embodiment, the electrode is gold and theoligonucleotide is attached to the electrode by a sulfur linkage. Inanother preferred embodiment the electrode is carbon and the linkage isa more stable amide bond. In either case, the linker connecting theoligonucleotide to the electrode is preferably comprised of 5 to 20 σbonds.

[0031] Yet another aspect of the invention relates to various methods ofdetection of the electrical current or charge generated by theelectrode-bound duplexes combined with an intercalative, redox-activespecies. In a preferred embodiment, the electrical current or charge isdetected using electronic methods, for example voltammetry oramperommetry, or optical methods, for example fluorescence orphosphoresence. In another preferred embodiment, the potential at whichthe electrical current is generated is detected by potentiommetry.

6. BRIEF DESCRIPTION OF DRAWINGS

[0032] Table 1 describes the electrochemical detection of single-basemismatches based on cyclic voltammograms measured for 1.0 μM daunomycinnoncovalently bound to duplex-modified electrodes.

[0033]FIG. 1 is a schematic diagram depicting DNA duplexes used forstudy of distance-dependent reduction of daunomycin. The right insertillustrates the daunomycin-guanine crosslink. The left insert shows thethiol-terminated tether which connects the duplex to the electrodesurface and provides 16 σ-bonds between the electrode and the basestack.

[0034]FIG. 2 illustrates cyclic voltammograms of gold electrodesmodified with daunomycin-crosslinked thiol-terminated duplexes (A)SH-^(5′)ATGGATCTCATCTAC+complement and (B)SH-^(5′)ATCCTACTCATGGAC+complement, where the bold Gs represent thedaunomycin crosslinking site.

[0035]FIG. 3 illustrates cyclic voltammograms of gold electrodesmodified with daunomycin-crosslinked thiol-terminated duplexescontaining TA and CA basepairs. The oligonucleotideSH-^(5′)ATTATATAATTGCT was hybridized with the corresponding complementscontaining either a T or a C opposite from the underlined A.

[0036]FIG. 4 describes the charges (Q_(c)) measured for daunomycin atDNA-modified electrodes containing different single-base mismatches. Toobtain the seven different mismatched duplexes the thiol-modifiedsequence, SH-^(5′)AGTACAGTCATCGCG, was hybridized with the followingseven different complements (the mismatch is indicated in bold, and thespecific basepair and the melting temperature of the duplex is given inparentheses): ^(5′)CGCGATGACTGTACT (TA, T_(m)=68° C.),^(5′)CGCGACGACTGTACT (CA, T_(m)=56° C.), ^(5′)CGCGATGTCTGTACT (TT,T_(m)=57° C.), ^(5′)CGCGATCACTGTACT (CC, T_(m)=56° C.),^(5′)CGCGATGGCTGTACT (GT, T_(m)=62° C.), ^(5′)CGCGATGAATGTACT (GA,T_(m)=60° C.), ^(5′)CGCGATGCCTGTACT (CT, T_(m)=58° C.).

[0037]FIG. 5 describes the charge obtained for DNA-modified electrodesin the presence of 1.0 μM daunomycin. the identified duplexes of varyingpercentages of GC content were either fully base-paired or contained asingle CA mismatch. Mismatch detection measuring the electrical currentor charge generated was independent of the sequence composition.

[0038]FIG. 6 describes the charges (Q_(c)) measured during the in situdetection of a CA mismatch. Electrodes were derivatized with thesequence SH-⁵ AGTACAGTCATCGCG, where either a C or a T was incorporatedinto the complement across from the underlined A. Using cyclicvoltammetry, the electrochemical response of daunomycin non-covalentlybound to duplex-modified electrodes was measured first for the intact TAor CA duplexes (TA vs. CA), secondly (after denaturation of the duplex)for the single stranded oligonucleotide (ss), thirdly (afterrehybridization with the opposite complement) again for the duplex (CAvs. TA), and lastly (after repeating the denaturation step) again forthe single-stranded oligonucleotide (ss).

[0039]FIG. 7 represents a schematic illustration of electrocatalyticreduction of ferricyanide. Methylene blue (MB⁺) is reducedelectrochemically through the DNA base stack to form leucomethylene blue(LB⁺). Ferricyanide is then reduced by LB⁺, causing the regeneration ofMB⁺ and the observation of catalytic currents.

[0040]FIG. 8 illustrates cyclic voltammograms of gold electrodesmodified with thiol-terminated duplexes containing TA and CA basepairsimmersed in a solution containing 1.0 μM methylene blue and 1.0 mMferricyanide. The oligonucleotide SH-^(5′)AGTACAGTCATCGCG was hybridizedwith the corresponding complements containing either a T or a C oppositefrom the underlined A.

7. DETAILED DESCRIPTION OF THE INVENTION

[0041] The expression “amplification of polynucleotides” includesmethods such as polymerase chain reaction (PCR), ligation amplification(or ligase chain reaction, LCR) and amplification methods based on theuse of Q-beta replicase. These methods are well known and widelypracticed in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202and Innis et al., 1990 (for PCR); and Wu et al., 1989a (for LCR).Reagents and hardware for conducting PCR are commercially available.Primers useful to amplify sequences from a particular gene region arepreferably complementary to, and hybridize specifically to sequences inthe target region or in its flanking regions. Nucleic acid sequencesgenerated by amplification may be sequenced directly. Alternatively theamplified sequence(s) may be cloned prior to sequence analysis. A methodfor the direct cloning and sequence analysis of enzymatically amplifiedgenomic segments has been described by Scharf (1986).

[0042] The term “base-stacking perturbations” refers to any event thatcauses a perturbation in base-stacking such as, for example, a base-pairmismatch, a protein binding to its recognition site, or any otherentities that form oligonucleotide adducts.

[0043] The term “denaturing” refers to the process by which strands ofoligonucleotide duplexes are no longer base-paired by hydrogen bondingand are separated into single-stranded molecules. Methods ofdenaturation are well known to those skilled in the art and includethermal denaturation and alkaline denaturation.

[0044] The term “hybridized” refers to two nucleic acid strandsassociated with each other which may or may not be fully base-paired.

[0045] The term “intercalative moieties” refers to planar aromatic orheteroaromatic moieties that are capable of partial insertion andstacking between adjacent base pairs of double-strandedoligonucleotides. These moieties may be small molecules or part of alarger entity, such as a protein. Within the context of this inventionthe intercalative moiety is able to generate a response or mediate acatalytic event.

[0046] The term “mismatches” refers to nucleic acid bases withinhybridized duplexes which are not 100% complementary. A mismatchincludes any incorrect pairing between the bases of two nucleotideslocated on complementary strands of DNA that are not the Watson-Crickbase-pairs A:T or G:C. The lack of total homology may be due todeletions, insertions, inversions, substitutions or frameshiftmutations.

[0047] The term “mutation” refers to a sequence rearrangement withinDNA. The most common single base mutations involve substitution of onepurine or pyrimidine for the other (e.g., A for G or C for T or viceversa), a type of mutation referred to as a “transition”. Other lessfrequent mutations include “transversions” in which a purine issubstituted for a pyrimidine, or vice versa, and “insertions” or“deletions”, respectively, where the addition or loss of a small number(1, 2 or 3) of nucleotides arises in one strand of a DNA duplex at somestage of the replication process. Such mutations are also known as“frameshift” mutations in the case of insertion/deletion of one of twonucleotides, due to their effects on translation of the genetic codeinto proteins. Mutations involving larger sequence rearrangement alsomay occur and can be important in medical genetics, but theiroccurrences are relatively rare compared to the classes summarizedabove.

[0048] The term “nucleoside” refers to a nitrogenous heterocyclic baselinked to a pentose sugar, either a ribose, deoxyribose, or derivativesor analogs thereof. The term “nucleotide” relates to a phosphoric acidester of a nucleoside comprising a nitrogenous heterocyclic base, apentose sugar, and one or more phosphate or other backbone forminggroups; it is the monomeric unit of an oligonucleotide. Nucleotide unitsmay include the common bases such as guanine (G), adenine (A), cytosine(C), thymine (T), or derivatives thereof. The pentose sugar may bedeoxyribose, ribose, or groups that substitute therefore.

[0049] The terms “nucleotide analog”, “modified base”, “base analog”, or“modified nucleoside” refer to moieties that function similarly to theirnaturally occurring counterparts but have been structurally modified.

[0050] The terms “oligonucleotide” or “nucleotide sequence” refers to aplurality of joined nucleotide units formed in a specific sequence fromnaturally occurring heterocyclic bases and pentofuranosyl equivalentgroups joined through phosphorodiester or other backbone forming groups.

[0051] The terms “oligonucleotide analogs” or “modifiedoligonucleotides” refer to compositions that function similarly tonatural oligonucleotides but have non-naturally occurring portions.Oligonucleotide analogs or modified oligonucleotides may have alteredsugar moieties, altered bases, both altered sugars and bases or alteredinter-sugar linkages, which are known for use in the art.

[0052] The terms “redox-active moiety” or “redox-active species” refersto a compound that can be oxidized and reduced, i.e. which contains oneor more chemical functions that accept and transfer electrons.

[0053] The term “redox protein” refers to proteins that bind electronsreversibly. The simplest redox proteins, in which no prosthetic group ispresent, are those that use reversible formation of a disulfide bondbetween to cysteine residues, as in thioredoxin. Most redox proteinshowever use prosthetic groups, such as flavins or NAD. Many use theability of iron or copper ions to exist in two different redox states.

[0054] The present invention provides a highly sensitive and accuratemethod based on an electrochemical assay using intercalative,redox-active species to determine the presence and location of a singleor multiple base-pair mismatches. Briefly, the system is comprised of(i) a reagent mixture comprising an electrode-bound oligonucleotideduplex to which an intercalative, redox-active moiety is associated and(ii) means for detecting and quantitating the generated electricalcurrent or charge as an indication for the presence of a fullybase-paired versus a mismatch containing duplex. The present inventionis particularly useful in the diagnosis of genetic diseases that arisefrom point mutations. For example, many cancers can be traced to pointmutations in kinases, growth factors, receptors binding proteins and/ornuclear proteins. Other diseases that arise from genetic disordersinclude cystic fibrosis, Bloom's syndrome, thalassemia and sickle celldisease. In addition, several specific genes associated with cancer,such as DCC, NF-1, RB, p53, erbA and the Wilm's tumor gene, as well asvarious oncogenes, such as abi, erbB, src, sis, ras, fos, myb and mychave already been identified and examined for specific mutations.

[0055] The present invention provides methods for detecting single ormultiple point mutations, wherein the oligonucleotide duplex carryingthe redox-active species is adsorbed and therefore continuously exposedto an electrode whose potential oscillates between a potentialsufficient to effect the reduction of said chemical moiety and apotential sufficient to effect the oxidation of the chemical moiety.This method is preferred over other methods for many reasons. Mostimportantly, this method allows the detection of one or more mismatchespresent within an oligonucleotide duplex based on a difference inelectrical current measured for the mismatch-containing versus the fullybase-paired duplex. Thus the method is based on the differences inbase-stacking of the mismatches and is independent of the sequencecomposition of the hybridized duplex, as opposed to existing methodsthat depend on thermodynamic differences in hybridization. Furthermore,this method is nonhazardous, inexpensive, and can be used in a widevariety of applications, alone or in combination with otherhybridization-dependent methods.

[0056] One particular aspect of the invention relates to the method forsequential detection of mismatches within a number of nucleic acidsamples which comprises the following steps. At least one strand of anucleic acid molecule is hybridized under suitable conditions with afirst nucleic acid target sequence forming a duplex which potentiallycontains a mismatch, and wherein one of the nucleic acids is derivatizedwith a functionalized linker. This duplex is then deposited onto anelectrode or an addressable multielectrode array forming a monolayer. Anintercalative, redox-active species (e.g., daunomycin) is noncovalentlyadsorbed (or crosslinked, if desired) onto this molecular lawn, and theelectrical current or charge generated is measured as an indication ofthe presence of a base pair mismatch within the adsorbed oligonucleotidecomplex. Subsequent treatment of the duplexes containing theintercalative, redox-active species under denaturing conditions allowsseparation of the complex, yielding a single-stranded monolayer ofoligonucleotides which can be rehybridized to a second oligonucleotidetarget sequence. The steps of duplex formation, adsorption of theintercalative, redox-active species, measurement of the electricalcurrent or charge, and denaturation of the complex to regenerate thesingle-stranded oligonucleotides may be repeated as often as desired todetect in a sequential manner genetic point mutations in a variety ofoligonucleotide probes.

[0057] The charges passed at each of the electrodes is measured andcompared to the wild-type, i.e. fully base-paired, sequences. Electrodeswith attenuated signals correspond to mutated sequences, while thosewhich exhibit no change in electrical current or charge are unmutated.Furthermore, the intensity of the signal compared to the wild-typesequence not only reports the presence of the mismatch but alsodescribes the location of the disruption within the analyzed duplex.

[0058] Another aspect of the invention relates to the method ofdetecting mutations utilizing electrocatalysis. Briefly, themodification of electrode surfaces with oligonucleotide duplexesprovides a medium that is impenetrable by negatively charged species dueto the repulsion by the high negative charge of oligonucleotides.However, electrons can be shuttled through the immobilized duplexes toredox-active intercalators localized on the solvent-exposed periphery ofthe monolayer, which in turn can catalytically reduce these negativelycharged species. More specifically, this electrocatalytic methodcomprises the following steps. At least one strand of a nucleic acidmolecule is hybridized under suitable conditions with a first nucleicacid target sequence forming a duplex which potentially contains amismatch, and wherein one of the nucleic acids is derivatized with afunctionalized linker. This duplex is then deposited onto an electrodeor a multielectrode array forming a monolayer. The assembly is immersedinto an aqueous solution containing both an intercalative, redox-activespecies (e.g., methylene blue) and a non-intercalative, redox-activespecies (e.g., ferricyanide). The electrical currents or chargescorresponding to the catalytic reduction of ferricyanide mediated bymethylene blue are measured for each nucleic acid-modified electrode andcompared to those obtained with wild-type, i.e. fully base-pairedsequences. Subsequent treatment of the duplexes under denaturingconditions allows separation of the complex, yielding a single-strandedmonolayer of oligonucleotides which can be rehybridized to a secondoligonucleotide target sequence. The steps of duplex formation,measurement of the catalytically enhanced electrical current or charge,and denaturation of the complex to regenerate the single-strandedoligonucleotides may be repeated as often as desired to detect in asequential manner genetic point mutations in a variety ofoligonucleotide probes. This particular method based on electrocatalysisat oligonucleotide-modified surfaces is extremely useful for systemswhere attenuated signals resulting from the presence of mismatches aresmall. The addition of a non-intercalative electron acceptor amplifiesthe signal intensity, and allows more accurate measurements. Thisapproach may be particularly useful to monitor assays based onredox-active proteins which bind to the oligonucleotide-modifiedsurface, but are not easily oxidized or reduced because the redox-activecenter is not intercalating.

[0059] The present invention further relates to the nature of theredox-active species. These species have a reduced state in which theycan accept electron(s) and an oxidized state in which they can donateelectron(s). The intercalative, redox-active species that are adsorbedor covalently linked to the oligonucleotide duplex include, but are notlimited to, intercalators and nucleic acid-binding proteins whichcontain a redox-active moiety.

[0060] An intercalator useful for the specified electrochemical assaysis an agent or moiety capable of partial insertion between stacked basepairs in the nucleic acid double helix. Examples of well-knownintercalators include, but are not limited to, phenanthridines (e.g.,ethidium), phenothiazines (e.g., methylene blue), phenazines (e.g.,phenazine methosulfate), acridines (e.g., quinacrine), anthraquinones(e.g., daunomycin), and metal complexes containing intercalating ligands(e.g., phi, chrysene, dppz). Some of these intercalators may interactsite-selectively with the oligonucleotide duplex. For example, thechrysene ligand is known to intercalate at the mispaired site of aduplex itself (Jackson, 1997), which can be exploited for selectivelocalization of an intercalator. This can be in particular useful toconstruct a duplex monolayer which contains the intercalative,redox-active species exclusively at its periphery.

[0061] In the case of redox-active nucleic acid-binding proteins,differences in DNA-mediated electron transfer between the duplex-boundprotein and the electrode allow for the detection of base-pairmismatches or other base-stacking perturbations. Examples ofredox-active proteins include, but are not limited to, mut Y,endonuclease III, as well as any redox-active cofactor-containingDNA-binding protein. Such proteins convert to one of an oxidized andreduced form upon reacting with a selected substrate, whereafter theoperation of the electrode regenerates the other of the oxidized andreduced forms.

[0062] The choice of a protein depends partially on its adsorption andbinding properties to biological macromolecules, i.e. nucleic acids, andwith non-biological macromolecules, whether in a homogeneous solution,or when immobilized on a surface. By changing the absorption or bindingcharacteristics, selectivity, signal to noise ratio and signal stabilityin these assays can be improved. The charge of a protein affects itsadsorption on surfaces, absorption in films, electrophoretic depositionon electrode surfaces, and interaction with macromolecules. It is,therefore, of importance in diagnostic and analytical systems utilizingproteins to tailor the charge of the protein so as to enhance itsadsorption or its binding to the macromolecule of choice, i.e thenucleic acid. In other cases, i.e when the detection assay is usedduring several cycles, it is of equal importance to be able tofacilitate desorption, removal, or stripping of the protein from themacromolecule. These assays require oligonucleotide duplexes that aredesigned such as to allow for site-specific binding of the protein ofchoice, which may require a single-stranded overhang. Once the proteinis adsorbed onto the nucleic acid, electrons are relayed via theoligonucleotide duplex to the electrode.

[0063] The nature of the non-intercalative, redox-active species used ina particular ectrocatalytic assay depends primarily on the redoxpotential of the intercalating, redox-active species utilized in thatsame assay. Examples include, but are not limited to, any neutral ornegatively charged probes, for example ferricyanide/ferrocyanide,ferrocene and derivatives thereof (e.g., dimethylaminomethyl-,monocarboxylic acid-, dicarboxylic acid-), hexacyanoruthenate, andhexacyanoosmate.

[0064] Yet another aspect of the invention relates to a method ofdetecting the presence or absence of a protein inducing base-stackingperturbations in DNA duplexes, this method comprising the followingsteps. At least one strand of a nucleic acid molecule is hybridizedunder suitable conditions with a second strand of nucleic acid moleculeforming a duplex, wherein one of the nucleic acids is derivatized with afunctionalized linker. This duplex is designed such to contain therecognition site of a protein of choice at a distinct site along thatduplex. This duplex is then deposited onto an electrode or anaddressable multielectrode array forming a monolayer and anintercalative, redox-active species is adsorbed onto this molecularlawn. In a preferred embodiment, the intercalative, redox-active speciesis site-specifically localized. In another preferred embodiment, theintercalative, redox-active species is crosslinked to theoligonucleotide duplex. This formed complex is then exposed to a samplesolution that potentially contains the specific protein and theelectrical current or charge generated is measured as an indication ofthe presence or absence of the protein. Subsequently, the protein isremoved under appropriate conditions to regenerate the duplex containingthe intercalative, redox-active moiety. The steps of duplex formation,adsorption or crosslinking of the intercalative, redox-active species,measurement of the electrical current or charge, and regeneration of theduplex containing the intercalative, redox-active moiety may be repeatedas often as desired to detect in a sequential manner the presence of aspecific protein in multiple sample solutions.

[0065] The charges passed at each of the electrodes are measured andcompared to the charges measured in a reference solution without theprotein. Electrodes with attenuated signals indicate the presence of theprotein in question which is binding to its recognition site, thuscausing a perturbation in base-stacking. Examples of proteins that canbe used for this assay include, but are not limited to, restrictionenzymes, TATA-binding proteins, and base-flipping enzymes (e.g., DNAmethylase).

[0066] The present invention also relates to the choice of nucleic acidprobes. Any nucleic acid, DNA or RNA, can be subjected to this mismatchdetection method, provided that the mismatch(es) to be detected liewithin the region between the attachment site of the intercalative,redox-active moiety and the electrode in order to be able to measure adifference in electrical current. The nucleic acid probes to be comparedmay comprise natural or synthetic sequences encoding up to the entiregenome of an organism. These probes can be obtained from any source, forexample, from plasmids, cloned DNA or RNA, or from natural DNA or RNAfrom any source, including bacteria, yeast, viruses, organelles andhigher organisms such as plants and animals. The samples may beextracted from tissue material or cells, including blood cells,amniocytes, bone marrow cells, cells obtained from a biopsy specimen andthe like, by a variety of techniques as described for example byManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Cold Spring Harbor, New York (1982), incorporatedherein by reference.

[0067] Alternatively, the sequences of choice can also be prepared bywell known synthetic procedures. For standard DNA and RNA synthesismethods, see for example “Synthesis and Applications of DNA and RNA” ed.S. A. Narang, Academic Press, 1987, M. J. Gait, “OligonucleotideSynthesis”, IRL Press, Wash. D.C. U.S.A., 1984, and “Oligonucleotidesand Analogues”ed. F. Eckstein, IRL Press, Wash. D.C. U.S.A., 1991, asincorporated herein by reference. Briefly, oligonucleotides andoligonucleotide analogs may be synthesized, conveniently through solidstate synthesis of known methodology. In a preferred embodiment, themonomeric units are added to a growing oligonucleotide chain which iscovalently immobilized to a solid support. Typically, the firstnucleotide is attached to the support through a cleavable linkage priorto the initiation of synthesis. Step-wise extension of theoligonucleotide chain is normally carried out in the 3′ to 5′ direction.When the synthesis is complete, the polymer is cleaved from the supportby hydrolyzing the linkage mentioned above and the nucleotide originallyattached to the support becomes the 3′ terminus of the resultingoligomer. Nucleic acid synthesizers such as the Applied Biosystems,Incorporated 380B are commercially available and their use is generallyunderstood by persons of ordinary skill in the art as being effective ingenerating nearly any oligonucleotide or oligonucleotide analog ofreasonable length which may be desired. Triester, phosphoramidite, orhydrogen phosphonate coupling chemistries are used with thesesynthesizers to provide the desired oligonucleotides or oligonucleotideanalogs.

[0068] In addition, the invention also relates to nucleic acid probesthat are constructed with a defined sequence comprised of nucleotide andnon-natural nucleotide monomers to restrict the number of binding sitesof the intercalative, redox-active agent to one single site. Forexample, in the case of the redox-active intercalator daunomycin mixednucleotide/non-natural nucleotide oligomers were prepared containing A-Tand/or I-C basepairs and one discrete guanine binding site to whichdaunomycin is crosslinked. The non-natural nucleotides are constructedin a step-wise fashion to produce a mixed nucleotide/non-naturalnucleotide polymer employing one of the current DNA synthesis methodswell known in the art, see for example “Synthesis and Applications ofDNA and RNA” ed. S. A. Narang, Academic Press, 1987, M. J. Gait,“Oligonucleotide Synthesis”, IRL Press, Wash. D.C. U.S.A., 1984, and“Oligonucleotides and Analogues” ed. F. Eckstein, IRL Press, Wash. D.C.U.S.A., 1991.

[0069] Methods and conditions used for contacting the oligonucleotidestrands of two DNAs, two RNAs or one DNA and one RNA molecule underhybridizing conditions are widely known in the art. Suitablehybridization conditions may be routinely determined by optimizationprocedures well known to those skilled in the art to establish protocolsfor use in a laboratory. See e.g., Ausubel et al., Current Protocols inMolecular Biology, Vol. 1-2, John Wiley & Sons (1989); Sambrook et al.,Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold SpringsHarbor Press (1989); and Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, NewYork (1982), all of which are corporated by reference herein. Forexample, conditions such as temperature, concentration of components,hybridization and washing times, buffer components, and their pH andionic strength may be varied.

[0070] Another aspect of the invention relates to a surface-modifiedelectrode and its use in a bioelectrochemical process, in whichelectrons are transferred directly between an electrode and anelectroactive biological material which is capable of accepting ordonating one or more electrons. Such a bioelectrochemical process can bein either direction. In particular, the invention provides electrodeshaving their surface modified with oligonucleotide duplexes carrying anintercalative, redox-active moiety. The electrode can be of any materialcompatible with the surface modifier being adsorbed or bound thereon,including, but not limited to noble metals such as gold, silver,platinum, palladium, as well as carbon. The preferred material for theelectrodes are gold and carbon.

[0071] The oligonucleotide duplex can be adsorbed onto the electrode inany convenient way. Preferably, the process of preparing such a modifiedelectrode comprises adsorbing the oligonucleotide duplex which isderivatized at the 5′-end with a functionalized linker chains onto theelectrode surface in one monolayer to obtain a uniform lawn. Theselinkers include, but are not limited to, thiol- or amine-terminatedchains. This process is generally understood by persons of ordinaryskill in the art as and is relatively simple, reproducible and caneasily be automated.

[0072] Furthermore, the density and composition of the monolayer issubject to variation depending on the selected assay. Methods ofdetecting single base-pair mismatches using intercalative, redox-activemoieties require a densely packed monolayer to prevent the adsorbedintercalative, redox-active moieties from diffusing into the lawn. Themethod for detecting the presence or absence of a protein requirespreferably an uneven monolayer comprised of duplexes of variable lengthto allow the protein to bind effectively to its recognition site alongthe duplex.

[0073] In addition, the present invention further relates to methods ofcreating a spatially addressable array of adsorbed duplexes. In apreferred embodiment, oligonucleotide duplexes of variable sequencecomposition that are derivatized at the 5′-end with a functionalizedlinker are deposited onto a multielectrode array. Subsequent treatmentof these electrode-bound duplexes under denaturing conditions yields amonolayer of single-stranded oligonucleotides, which can then behybridized with a complementary oligonucleotide probe that potentiallycontains a mismatch. In another preferred embodiment, shortoligonucleotide duplexes (5 to 10 base-pairs in length) that arederivatized on one end with a functionalized linker and containsingle-stranded overhangs (5 to 10 nucleotides in length) of designedsequence composition at the opposite end are deposited onto amultielectrode array to generate a spatially addressable matrix. Theseelectrode-bound duplexes can then be hybridized with single-stranded ordouble-stranded oligonucleotides that contain the complementaryoverhang.

[0074] Solid supports containing immobilized molecules have beenextensively used in research, in clinical analyses and in the commercialproduction of foods and chemicals (see e.g., U.S. Pat. No. 5,153,166;Akashi, 1992). Immobilized nucleic acids are used in hybridizationassays (Lamture, 1994) and immobilized proteins in radioimmuno or ELISAassays (see, U.S. Pat. No. 5,314,830). In addition, enzymes have beenimmobilized to facilitate their separation from product and to allow fortheir efficient and repetitive use. A number of important factors haveto be considered in the development of an effective immobilizationprocedure. First, the procedure must minimize non-specific adsorption ofmolecules. Second, the procedure must maintain the functional integrityof the immobilized molecules. Third, the stability of the bond betweenthe support and the immobilized molecule must be such to avoid leachingwhich would lead to reduced accuracy and sensitivity. Finally, thecoupling procedure must be efficient enough to result in a support witha high capacity for the target molecules as well as be cost effective.

[0075] Another aspect of the invention relates to measuring theelectrical current as a function of degree of hybridization of theoligonucleotide duplex adsorbed onto the electrode. When theintercalative, redox-active species is exposed to electrochemical orchemical energy, the electrical current may be continuously detectedusing techniques well known in the art. These include, but are notlimited electronic methods, for example voltammetry or amperommetry, oroptical methods, for example fluorescence or phosphoresence.

[0076] Generally, photoluminescence excitation and emission occur withelectromagnetic radiation of between about 200 nanometers and about 900nanometers in wavelength. Likewise, chemiluminescent andelectrochemiluminescent emission generally occur with the emittedelectromagnetic radiation being between about 200 nanometers and about900 nanometers in wavelength. The potential at which the reduction oroxidation of the chemical moiety occurs depends upon its exact chemicalstructure as well as factors such as the pH of the solution and thenature of the electrode used. It is well known how to determine theoptimal emission and excitation wavelengths in a photoluminescent systemand the optimal potential and emission wavelength of anelectrochemiluminescent and chemiluminescent system.

[0077] There are many methods for quantifying the amount of luminescentspecies present. The rate of energy input into the system can provide ameasure of the luminescent species. Suitable measurements include, forexample, measurements of electric current when the luminescent speciesis generated electrochemically, the rate of reductant or oxidantutilization when the luminescent species is generated chemically or theabsorption of electromagnetic energy in photoluminescent techniques. Inaddition, the luminescent species can be detected by measuring theemitted electromagnetic radiation. All of these measurements can be madeeither as continuous, event-based measurements, or as cumulative methodswhich add the signal over a long period of time. Event-basedmeasurements may be carried out with photomultiplier tubes, photodiodesor phototransistors to produce electric currents proportional inmagnitude to the incident light intensity, or by using charge coupledevices. Examples of cumulative methods are the integration ofevent-based data, and the use of photographic film to provide cumulativedata directly.

[0078] The publications and other reference materials referred to hereindescribe the background of the invention and provide additional detailregarding its practice and are hereby incorporated by reference. Forconvenience, the reference materials are referenced and grouped in theappended bibliography.

[0079] The present invention is further described in the followingexamples. These examples are for illustrative purposes only, and are notto be construed as limiting the scope of the invention as set forth inthe appended claims.

EXAMPLES

[0080] Materials. Phosphoramidite reagents (including the C₆S-S thiolmodifier) were obtained from Glen Research. [γ-³²P]dATP was obtainedfrom NEN-DuPont. Potassium ferrocyanide (Fisher) was recrystallized fromaqueous solution prior to use. Daunomycin was obtained from Fluka.

[0081] Synthesis of Derivatized Duplexes. Oligonucleotides immobilizedon a controlled pore glass resin were treated in succession withcarbonyldiimidazole and 1,6-diaminohexane (1 g/10 ml dioxane, 30min/ea.) at the 5′-hydroxy terminus before cleavage from the resin(Wachter, 1986). After deprotection, the free amine was treated with2-pyridylthiopropionic acid N-succinimide ester to produce a disulfide(Harrison, 1997). The sequences were purified by reverse-phase HPLC,converted to free thiols using dithiothreitol, and repurified beforehybridization to their complements. Derivatized oligonucleotides werecharacterized by mass-assisted laser desorption ionizationtime-of-flight mass spectrometry and HPLC retention times. Duplexes werehybridized in deoxygenated 5 mM phosphate/50 mM NaCl (pH 7) by heatingto 90° C. followed by slow cooling to room temperature. Unprotectedduplexes were stored frozen under argon to prevention oxidation of thethiol.

[0082] Atomic Force Microscopy (AFM). All AFM images were collectedusing a MultiMode AFM running on the NanoScope IIIa controller (DigitalInstruments, Santa Barbara, Calif.). A glass AFM chamber (DigitalInstruments, Santa Barbara, Calif.) and a fluid volume of approximately50 microliters were used for the experiments. Si₃N₄ cantilevers (springconstant, 0.06 N/m) with integrated, end-mounted oxide-sharpened Si₃N₄probe tips were used. The applied vertical force of the AFM probe duringimaging was minimized to beneath 100 pN. Continually adjusting thecantilever deflection feedback setpoint compensated for thermal driftingof the cantilever and a consistent, minimum force was maintained AFMheight calibrations were carried out on a NIST-traceable 180-nm heightstandard and then confirmed by measuring a single-atom step in the Augold surface. The AFM images were recorded in “Height” (or constantforce) mode. Holes in the monolayer used to determine monolayerthicknesses were prepared by decreasing the scan size to approximately100-150 nm, increasing the scan rate to 24-30 Hz, and increasing thevertical force by advancing the setpoint several units. After about oneminute, the scan size, scan rate, and setpoint were returned to theirprevious values, and images featuring a bare gold square were captured.All images captured for height-contrast analysis were recorded atminimum vertical tip forces. This was accomplished by decreasing theset-point until the tip disengaged from the surface, then reintroducingit with the minimum force required to achieve a stable image. In severalcases, the film height was also measured in tapping mode, and gave thesame result as the contact-mode experiments.

[0083] Electrochemistry. Cyclic voltammetry (CV) was carried out on 0.02cm² polycrystalline gold electrodes using a Bioanalytical Systems (BAS)Model CV-50W electrochemical analyzer at 20±2° C. in 100 mM phosphatebuffer (pH 7). A normal three-electrode configuration consisting of amodified gold-disk working electrode, a saturated calomel referenceelectrode (SCE, Fisher Scientific), and a platinum wire auxiliaryelectrode was used. The working compartment of the electrochemical cellwas separated from the reference compartment by a modified Luggincapillary. Potentials are reported versus SCE. Heterogeneouselectron-transfer rates were determined and analyzed by CV (Nahir, 1994;Weber, 1994; Tender, 1994).

[0084] Ellipsometry. Optical ellipsometry (λ=632.8 nm) was carried outon dried samples at 25° C. using a Gaertner Model L116C ellipsometer.

Example 1 Site-specific Incorporation of a Redox-Active Intercalatorinto a DNA Duplex

[0085] The redox-active intercalator daunomycin (DM) (Arcamone, 1981)was incorporated into the DNA duplex to investigate charge transductionthrough these duplexes (FIG. 1). DM undergoes a reversible reduction(Molinier-Jumel, 1978; Berg, 1981) within the potential window of themonolayers (Kelley, 1997a), and covalent adducts of intercalated DMcrosslinked to the 2-amino group of guanine (Leng, 1996) have beencrystallographically characterized within duplex DNA (Wang, 1991). Thus,a series of oligonucleotides primarily containing A-T or inosine (I)-Cpairs were constructed with discrete guanine binding sites to which DMwas crosslinked. Preferably, thiol-terminated duplexes (0.1 mM)containing an adjacent pair of guanines were hybridized, incubated with0.2% formaldehyde and 0.2 mM DM in 5 mM phosphate, 50 mM NaCl, pH 7 for1 h, and phenol extracted to remove excess DM.

[0086] Moving the guanine site along the duplex resulted in a systematicvariation of the through-helix DM/gold separation, and allowed aninvestigation of the effect of distance on the dynamics of chargetransport through the monolayers (FIG. 1).

Example 2 Characterization of DNA Duplexes Modified with a Redox-ActiveIntercalator

[0087] Modified duplexes were characterized by mass spectrometry,ultraviolet/visible absorption spectroscopy, and thermal denaturationexperiments, all of which were consistent with a 1:1 DM-duplexstoichiometry. For example, the duplexSH—(CH₂)CONH(CH₂)₆NHCO₂-^(5′)ATCCTACTCATGGAC with its inosine complementmodified with DM was analyzed by MALDI-TOF spectrometry. Mass-to-chargeratios (found/calc) of 5284/(5282) (DM+SH strand), 4541/(4540)(complement), and 4742/(4742) (SH strand) were detected. These valuescorrespond to the calculated masses for fragments expected from thisduplex. UV-visible absorption spectroscopy also revealed a 1:1 duplex/DMstoichiometry based upon comparison of the duplex absorbance at 260 nm(M=14.9×10³ M⁻¹cm⁻¹) and the absorbance of intercalated DM at 480 n(M=5.1×10³ M⁻¹ cm⁻¹. In the presence of 100 mM phosphate, 100 mM MgCl₂,and at pH 7, thermal denaturation studies of 5 DM duplexes (monitored byabsorbance at 260 nm) revealed melting temperatures of 48 and 50° C. forthe native and daunomycin-crosslinked duplexes, respectively. A similarmelting profile was obtained by monitoring hypochromicity at 482 nm forthe DM duplex.

Example 3 Preparation of Gold Electrodes Derivatized with DNA Duplexes

[0088] Electrodes were conveniently prepared by modifying gold surfaceswith 15 base-pair DNA duplexes derivatized at the 5′ end with athiol-terminated alkane chain. Bulk gold electrodes were polishedsuccessively with 0.3- and 0.5-μM alumina (Buhler), sonicated for 30min, and etched in 1.0 M sulfuric acid. Au(111) surfaces were preparedby vapor deposition onto mica or glass (Widrig, 1991; Zei, 1983).Electrodes were then modified by incubation in 0.1 mM solutions ofderivatized DNA duplexes in 5 mM phosphate/50 mM NaCl (pH 7) for 12-48 hat ambient temperature. Modified electrodes were rinsed in buffer priorto use.

[0089] Before deposition of the duplexes onto the gold surfaces, thepresence of the free thiols was confirmed using a spectroscopic assaybased on dithionitrobenzene (Riddles, 1979). Subsequently, the sampleswere deposited onto the gold surfaces for 12-24 h.

[0090] Electrochemical assays, radioactive tagging experiments, andatomic force microscopy (AFM) (Kelley, 1997a, 1997b) all indicate thatthe oligonucleotides form densely packed monolayers oriented in anupright position with respect to the gold surface.

Example 4 Characterization of Modified DNA Duplexes Monolayers on GoldSurfaces

[0091] The DM-modified duplexes readily formed self-assembled monolayerson gold. AFM studies of modified films reveal densely packed monolayerswith heights greater than 45 Å at open circuit. More specific, AFMstudies were carried out under electrochemical control, and revealedthat the DNA films undergo a potential-dependent change in structure. Atopen circuit, the monolayer film height is 45(3) Å. Based on theanisotropic dimensions of the 15-base pair duplexes (20 Å in diametervs. 60 Å in length), this thickness indicates that the helical axis isoriented ˜45″ from the gold surface. At applied voltages negative of thepotential of zero charge, film thickness of ˜60 Å are observed; morepositive potential cause a drop in the film height to a limiting valueof 20 Å at low surface coverages.

[0092] Based on the crossectional area of DNA (˜3 nm²) and thegeometrical area of the gold electrodes (0.02 cm²), the maximum surfacecoverage of DNA was calculated as ˜6×10⁻¹¹ mol/cm. Coulometry atelectrodes modified with duplexes containing crosslinked DM revealed aDM surface coverage of 7.5(7)×10⁻¹¹ mol/cm ², indicating that thesurface is densely packed with the modified duplexes. The DM valueappeared to exceed slightly the theoretical Υ for DNA, and likelyresulted from additional electrode surface roughness.

[0093] To assess routinely the surface coverage of DM-derivatized DNA ongold, the electrochemical response of Fe(CN)₆ ⁴⁻ (2 mM) was monitored.This negatively charged ion is repelled from the modified-electrodesurface by the polyanionic DNA, and exhibits essentially no responsewhen the surface is well covered. While not a direct measure of surfacecoverage, this technique allowed the convenient assay of individualelectrodes for adequate modification.

[0094] Cyclic voltammograms of these surfaces showed the reversiblereduction of DM at −0.65 V versus SCE (Molinier-Jumel, 1978; Berg,1981). These films were extremely stable and exhibited responsescharacteristic of surface-bound species (e.g., linear plots of peakcurrent versus scan rate) (Bard, 1980).

Example 5 Measurement of Electrochemical Response of a Redox-ActiveIntercalator Crosslinked to a Fully Base-Paired DNA Duplex on a GoldSurface

[0095] Integration of the electrochemical response yielded a surfacecoverage (Υ) of electroactive daunomycin of 7.5(7)×10⁻¹¹ mol/cm², avalue in good agreement with the coverages of 15-base pair duplexespreviously measured via ³²P labeling (Kelley, 1997a). However,significant fluctuations in the surface coverages of DM-modifiedduplexes were observed. Therefore, only electrodes which exhibited bothlarge integrated currents for the reduction of crosslinked DM and anattenuated responses for the oxidation of ferrocyanide in solution werestudied.

[0096] Given the 1:1 stoichiometry of crosslinked DM to DNA, theobserved data indicated that all of the bound DM was electrochemicallyreduced. Doping these films with increasing percentages of DM-freeduplexes resulted in a linear decrease in the observed electrochemicalsignals (as determined from coulometric assays), consistent with each ofthe bound intercalators being electrochemically active.

[0097] Remarkably, efficient reduction of DM was observed regardless ofits position along the 15-base-pair sequence as illustrated in FIG. 2.Based on molecular modeling, the DM/gold separations span ˜25 Å. Thethrough-helix DM-electrode separation is >10 Å for DM bound at the endof the duplex closest to the electrode (FIG. 2A), and the DM-electrodeseparation is >35 Å (FIG. 2B) for DM crosslinked to the end of theduplex farthest from the electrode. The surface coverage ofelectroactive daunomycin for these 15 base-pair duplexes as measured byintegrating the currents within the illustrated voltammograms were0.65×10⁻¹⁰ mol/cm² and 0.80×10⁻¹⁰ mol/cm², respectively. The DM:DNAstoichiometry for these same samples, measured by absorptionspectroscopy were 0.9:1 and 1.1:1, respectively. Thus, the charge didnot depend on distance, but did reflect the yield of crosslinking.

Example 6 Measurement of Electrochemical Response of a Redox-ActiveIntercalator Crosslinked to a Mismatch-Containing DNA Duplex on a GoldSurface

[0098] Electrochemical responses of a redox-active intercalatorcrosslinked to a mismatch-containing DNA duplex on a gold surface weremeasured to determine whether these observed rates were a result ofdirect contact between the redox-active cofactor and the electrodesurface (which has previously been shown to yield apparentlydistance-independent heterogeneous electron transfer (Feng, 1995,1997)). A single site within the 15-base-pair duplex was mutated toproduce a CA mismatch (known to cause local disruptions in the DNA basestack (Patel, 1984; Aboul-ela, 1985) between the intercalated DM and theelectrode surface. FIG. 3 illustrates that such a simple changevirtually eliminated the electrochemical response.

[0099] The coulometry of DM at electrodes modified with CA-containingduplexes varied to some degree as a function of the surface coverage. Athigh surface coverages (as determined by the ferrocyanide assay),essentially no signal was observed with the mismatched duplexes.However, at more moderate surface coverages, small signals correspondingto the reduction of DM were found. These typically did not exceed 30% ofthe signals found for the TA duplexes. The morphology of partial DNAmonolayers is unknown.

[0100] Significantly, sequences in which the positions of the DM and CAmismatch were reversed (such that the mismatch was located above the DMrelative to the gold) showed no diminution in the electrochemicalresponse. AFM images of the CA-mutated sequences were identical to thoseof the TA analogs (monolayer thicknesses of ˜40 Å at open circuit),revealing that the bulk structure of the DNA films was not significantlyaltered by the presence of a mismatch. Moreover, the oxidation offerrocyanide was similarly attenuated at both surfaces. Expected massesfor DM-crosslinked DNA duplexes (accounting for the single base change)were measured by mass spectrometry, and spectrophotometric assaysrevealed that the extent of crosslinking was identical in both fullypaired and mismatched sequences.

[0101] The exquisite sensitivity of the electrochemistry of DM tointervening lesions in the base stack provides therefore the basis foran exceptionally versatile DNA-mismatch sensor.

Example 7 Analysis of the Electrochemical Behavior of Fully Base-Pairedor Mismatch-Containing DNA Duplexes Containing Non-CrosslinkedIntercalators

[0102] A practical method to detect mismatches utilizes a system basedon non-crosslinked, intercalative, redox-active species. Theelectrochemistry of DM non-covalently intercalated into DNA-modifiedfilms was studied in order to develop a general approach to testheterogeneous sequences that may possess more than one guanine-bindingsite. Coulometric titrations confirmed that DM strongly binds tosurfaces modified with fully base-paired duplexes, and yielded affinityconstants very similar to those determined for homogeneous solutions(Arcamone, 1981; Molinier-Jumal, 1978; Berg, 1981). At bulk DMconcentrations≧1 μM, the modified electrodes were saturated withintercalator, and hold approximately one intercalator persurface-confined duplex. Furthermore, intercalators non-covalently boundto these films exhibited electrochemical properties quite similar tothose described for crosslinked DM, with the exception that the bindingwas reversible, i.e. in pure buffer solutions, decreasing voltammetricsignals were observed until total dissociation was evident.

[0103] In accord with the studies of covalently bound DM, incorporationof a single CA mismatch into these duplexes dramatically decreased theelectrochemical response (Table 1). The magnitude of this mismatcheffect depended strongly on the location of the CA base step along thesequence: when the mutation was buried deep within the monolayer, themeasured charge drops by a factor-of 3.5(5) (relative to theWatson-Crick duplex), but by only 2.3(4) when it was located near thesolvent-exposed terminus. These observations were consistent with DMoccupying sites near the top of the densely packed monolayer, assuggested in earlier studies of methylene blue bound to these samesurfaces (Kelley, 1997b). The intensity of the electrochemical signalstherefore not only reports the presence of the mismatch but also maydescribe the location of the disruption.

[0104] In addition, lateral charge diffusion within these monolayers wasanalyzed. For example, a series of fully base-paired films (sequence:SH-^(5′)AGTACAGTCATCGCG) doped with increasing fractions ofCA-mismatched helices were prepared (the mismatch was localized at thebase step denoted by the bold C in the above sequence.) The coulometricresponse of DM non-covalently bound to these surfaces was stronglydependent on the film composition such that the electrochemical signalsdecreased linearly with increasing percentages of mutated duplexes. Asthere is no measurable difference in the affinities of DM toward TA-versus CA-containing films, this linear response indicated that theelectroinactive intercalators (presumably those molecules bound tomutated helices) are not reduced by lateral charge transfer from theelectroactive species. This result further supports a through-helixpathway for charge transduction, as intermolecular interactions betweenintercalators bound to different duplexes in the film evidently do notmediate efficient electron transfer.

Example 8 Analysis of Mutation Dependence of Electrochemical Response

[0105] To explore the scope of this mismatch detection strategy, thecharge (Q_(c)) for DM at DNA-modified electrodes containing differentsingle-base mismatches was analyzed (FIG. 4). The seven differentmismatched duplexes were obtained by hybridization of the thiol-modifiedsequence, SH-⁵ AGTACAGTCATCGCG, with the following seven differentcomplements (the mismatch is indicated in bold, and the specificbasepair and the melting temperature of the duplex is given inparentheses): ^(5′)CGCGATGACTGTACT (TA, T_(m)=68° C.),^(5′)CGCGACGACTGTACT (CA, T_(m)=56° C.), ^(5′)CGCGATGTCTGTACT (TT,T_(m)=57° C.), ^(5′)CGCGATCACTGTACT (CC, T_(m)=56° C.),^(5′)CGCGATGGCTGTACT (GT, T_(m)=62° C.), ^(5′)CGCGATGAATGTACT (GA,T_(m)=60° C.), ^(5′)CGCGATGCCTGTACT (CT, T_(m)=58° C.). The charges werethen calculated by integrating background-subtracted cyclicvoltammograms. The obtained values were based on >5 trials, and theresults were comparable for experiments run side-by-side or utilizingdifferent sample preparations. The melting temperatures of the oligomersin solution were measured by monitoring duplex hypochromicity at 260 nmusing samples that contained 10 μM duplex, 100 mM MgCl₂, and 100 mMphosphate at pH 7.

[0106] Coulometric analysis confirmed that the attenuation of thecharacteristic DM response was strongly dependent upon the identity ofthe mutation. In general, pyrimidine-pyrimidine and purine-pyrimidinemismatches caused marked decreases in the electrochemical signals; theone purine-purine pair studied (a GA mismatch, which is notoriouslywell-stacked within duplex DNA (Patel, 1984; Aboul-ela, 1985)) did notshow a measurable effect. Surprisingly, a significant decrease wascaused by a GT pair, which is also not highly disruptive to the helix.This wobble base pair, although thermodynamically stable, appears tomediate electron transfer poorly.

[0107]FIG. 4 illustrates that across a very narrow range of duplexthermal stabilities, large differences in the electrochemical responsewere observed. Overall, the electrochemical properties of filmscontaining the different mismatches correlated with the degree ofdisruption to base stacking with the individual duplexes. These resultsunderscore the sensitivity of this electrochemical assay to basestacking within DNA, and demonstrate the viability of detectingmismatches based upon charge transduction through thin films.

Example 9 Analysis of Sequence Dependence of Mismatch Detection Assay

[0108] A single CA mismatch was incorporated into three different DNAduplexes to test for the sequence dependence of the assay. The duplexesfeatured varying percentages of GC content, representing a wide range ofduplex stabilities. The melting temperatures for these duplexes, asdetermined by thermal denaturation measurements obtained by monitoringhypochromicity at 260 nm in duplex solutions containing 10 μM duplex,100 mM phosphate, and 100 mM MgCl₂ were: (SH-^(5′)-ATATAATATATGGAT):TA=47° C., CA=32° C.; (SH-^(5′)-AGTACAGTCATCGCG): TA=68° C., CA=56° C.;(SH-^(5′)-GGCGCCCGGCGCCGG): GC=82° C., CA=69° C. The charge wasquantitated from integrating background-subtracted cyclic voltammogramsobtained at υ=100 mV/s and was corrected for electrode area. Asillustrated in FIG. 5, the characteristic drop in coulometric signalsfor DNA duplexes containing a single CA mismatch compared to fullybase-paired DNA films was essentially invariant across AT-rich toGC-rich sequences. This sequence-independent response is not achievableusing traditional mismatch detection assays based upon differentialhybridization.

Example 10 Analysis of Electrochemical Response During Repeated Cycles

[0109] To extend this methodology to single-stranded targets, techniquesfor in situ hybridization were developed. Thiol-modified duplexes weredeposited on the gold surface, heat denatured, thoroughly rinsed, thenrehybridized with the desired target by incubation in ≧50 pmol ofsingle-stranded oligonucleotide. The electrochemical properties of theresulting surfaces were identical to those described above, suggestingthe suitability of this system for genomic testing.

[0110] For example, a 15-base-pair oligonucleotide,^(5′)AGTACAGTCATCGCG, which was derivatized with a thiol-terminatedlinker, was hybridized both to its native complement and to a mutatedcomplement (at the site underlined in the sequence), generating a fullybase-paired duplex and a CA mismatch-containing duplex, respectively(FIG. 6). These duplexes were deposited on separate electrodes and theelectrochemical responses of DM non-covalently bound to these duplexeswere measured using cyclic voltammetry (υ=100 mV/s, 1.0 μM DM). FIG. 6illustrates that DM exhibited electrochemical responses characteristicof fully base-paired and CA-mutated films, respectively. The surfaceswere then denatured by immersing the electrodes in 90° C. pure bufferfor 2 min to yield single-stranded monolayers of identical sequence.Cyclic voltammetry of DM at these electrodes now revealed nearlyidentical responses, with the reduction appearing highly irreversible,broadened, and becoming smaller as a function of increasing scans.Importantly, the electrode that initially possessed the CA mismatchdisplayed a large signal (for the first scan) after denaturation, whilethe reverse was true for the corresponding TA analog. New duplexes wereformed by incubating the electrodes with 100 pmol of the oppositecomplement in the presence of buffered 100 mM MgCl₂ such that thecomplements were traded (TA→CA, CA→TA), and the electrochemistry at theduplex-modified films again showed the characteristic behavior expectedfor fully base-paired and CA-mutated films. Finally, the electrodes wereagain heated to denature the duplexes and quantitation of the responseshowed again the characteristics for single-stranded oligonucleotides.Thus, electrodes can be cycled through this sequence of eventsrepeatedly, indicating a practical means to detect point mutationswithin natural DNAs.

Example 11 Detection of Genetic Mutations Within a Specific Region ofthe p53 Gene Using Direct Current Measurement of Thiol-Modified Duplexeson Gold Surfaces

[0111] A specific embodiment utilizes a gold-microelectrode array withapproximately thirty addressable sites. A different 20-base pair duplexderivatized with a hexylthiol linker is attached to each of these sitesby deposition form a concentrated duplex solution overnight. Thesequences are chosen to correspond to the 600-base pair region withinexons 5 through 8 of the p53 gene where most of the cancer-relatedmutations are found. The array is immersed in aqueous solution at 90° C.for 60 seconds to denature the immobilized duplexes and remove thecomplementary strands. The human sample containing the p53 gene isfragmented either before or after amplification. A solution containingthe fragmented genomic single-stranded DNA is deposited on the array forone hour to allow hybridization to occur. Then, in the presence of a 1.0μM DM solution, the charge passed at each of the electrodes is measured,and the response for each sequence is compared to that obtained from thewild-type (i.e. fully base-paired) sequences. Electrodes with attenuatedsignals correspond to mutated subsequences, while those which exhibitedthe expected charge are classified as unmutated.

Example 12 Detection of Mutations Using Electrocatalytic CurrentsGenerated at DNA-Modified Surfaces

[0112] The signals corresponding to mismatched and fully-pairedsequences can be more highly differentiated by monitoring catalyticcurrents at DNA-modified surfaces. Electrons can be shuttled through theimmobilized duplexes to redox-active intercalators localized on thesolvent-exposed periphery of the monolayer, and then negatively-chargedsolution-borne species (which are electrostatically prohibited from theinterior of the monolayer) are catalytically reduced by theintercalating mediators. Since the catalytic reaction essentiallyamplifies the signal corresponding to the intercalator, the attenuationof this response in the presence of the mismatch is significantly morepronounced. In a specific embodiment, the sequenceSH-^(5′)AGTACAGTCATCGCG was deposited on an electrode both hybridizedwith a fully base-paired complement, and with a complement containing aCA mismatch (the position of the mismatch is denoted in bold). Theseduplexes were immersed in a solution containing 1.0 μM methylene blueand 1 mM ferricyanide. In the presence of either of these reagentsalone, only small direct currents were measured. However, in thepresence of a mixture of the intercalator and the negatively chargedprobe, pronounced currents were measured corresponding to theelectrocatalytic reduction of ferricyanide by methylene blue. The amountof current observed for the TA and CA containing films differdramatically; using electrocatalysis, the mismatched duplex can bedifferentiated from the fully base-paired duplexes by a factor ofapproximately 100. Moreover, as illustrated in FIG. 8, the peakpotentials for the TA and CA duplexes are significantly separated,allowing the presence of the mismatch to be detected potentiometrically.This approach therefore represents an extremely sensitive means todetect genetic mutations electrochemically.

Example 13 Detection of Genetic Mutations Within a Specific Region ofthe p53 Gene Using Electrocatalytic Current Measurement ofThiol-Modified Duplexes on Gold Surfaces

[0113] Another specific embodiment involves detecting the mutationswithin the p53 gene using electrocatalysis. A different 20-base pairduplex derivatized with a hexylthiol linker is attached to each ofapproximately thirty addressable sites of a gold-microelectrode array bydeposition form a concentrated duplex solution overnight. The sequencesare chosen to correspond to the 600-base pair region within exons 5through 8 of the p53 gene where most of the cancer-related mutations arefound. The array is immersed in aqueous solution at 90° C. for 60seconds to denature the immobilized duplexes and remove thecomplementary strands. The human sample containing the p53 gene isfragmented either before or after amplification. A solution containingthe fragmented genomic single-stranded DNA is deposited on the array forone hour to allow hybridization to occur. The array is rinsed andsubmerged in a solution containing 1.0 μM methylene blue and 1.0 mMferricyanide. The pronounced currents that are observed result from theelectrocatalytic reduction of the solution-borne ferricyanide bymethylene blue adsorbed at the solvent-exposed duplex sites. Thesecatalytic currents are measured for each addressable electrode andcompared with those obtained with the wild-type sequence to detectpotential sites of mutations.

Example 14 Detection of Genetic Mutations Within a Gene of InterestUsing Direct or Electrocatalytic Current Measurement of Amine-ModifiedDuplexes on Carbon Surfaces

[0114] Another embodiment utilizes a carbon electrode. The electrode isoxidized at +1.5 V (vs. Ag/AgCl) in the presence of K₂Cr₂O₇ and HNO₃,and treated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Duplexescorresponding to mutated sequences of a specific gene of interest arederivatized with a hexylamine linker and applied to the electrodesurface. The device is immersed in aqueous solution at 90° C. for 60seconds to, generate a single-stranded monolayer, and the fragmentedgenomic DNA sample is hybridized to the immobilized probes at roomtemperature for 1 hour. The detection of mutations is accomplished by(i) measuring direct currents in the presence of 1.0 μM daunomycinsolution, or (ii) by measuring catalytic currents in the presence of 1.0μM methylene blue and 1.0 mM ferricyanide. Charges passed at eachelectrode are measured, and the response for each sequence is comparedto that obtained with wild-type, i.e. fully base-paired, sequences.Attenuated signals correspond to mutated subsequences, while those whichexhibit no change in current are classified unmutated.

[0115] Although the invention has been described with reference toparticular applications, the principles involved may be used in otherapplications which will be apparent to those skilled in the art. Theinvention is accordingly to be limited only by the scope of the claimswhich follow. TABLE I Electrochemical Detection of Single-BaseMismatches^(a) Q_(c(int)) (nC)^(b) T_(m) (° C.)^(c)SH-^(5′)AGTACAGTCATCGCG 165(37)  68 TCATGTCAGTAGCGCSH-^(5′)AGTACAGTCATCGCG 56(15) 56 TCATGTCAGCAGCGCSH-^(5′)AGTACAGTCATCGCG 95(18) 57 TCATGTCTGTAGCGCSH-^(5′)AGTACAGTCATCGCG 51(23) 56 TCATGTCACTAGCGCSH-^(5′)AGTACAGTCATCGCG 49(30) 62 TCATGTCGGTAGCGCSH-^(5′)AGTACAGTCATCGCG 153(38)  60 TCATGTAAGTAGCGCSH-^(5′)AGTACAGTCATCGCG 93(17) 58 TCATGTCCGTAGCGC

What is claimed is:
 1. A method of detecting one or more base-stackingperturbations in a target sequence comprising: (a) hybridizing a firstsingle stranded nucleic acid to a second single stranded nucleic acid toform a first complex; (b) depositing said first complex onto anelectrode or an addressable multielectorde array; (c) adding anintercalative, redox-active moiety to said first complex to form asecond complex; and (d) measuring an electron transfer event betweensaid electrode or addressable multielectrode array and saidintercalative, redox-active moiety as an indication for the presence orabsence of said base-stacking perturbations.
 2. A method according toclaim 1, wherein said base-stacking perturbations are point mutations,protein-DNA adducts, adducts between any chemical entity and said targetsequence, or combinations thereof.
 3. A method according to claim 1,wherein said intercalative, redox-active moiety is either noncovalentlyadsorbed or crosslinked to said first complex.
 4. A method according toclaim 1, wherein said intercalative, redox-active moiety is anintercalator.
 5. A method according to claim 1, wherein saidintercalative, redox-active moiety is an intercalator selected from thegroup consisting of phenanthridines, phenothiazines, phenazines,acridines, and anthraquinones.
 6. A method according to claim 1, whereinsaid intercalative, redox-active moiety is daunomycin.
 7. A methodaccording to claim 1, wherein said intercalative, redox-active moiety isa part of a protein.
 8. A method according to claim 1, wherein saidintercalative, redox-active moiety is mut Y.
 9. A method according toclaim 1, wherein said electrode or addressable multielectrode array isgold.
 10. A method according to claim 1, wherein said electrode oraddressable multielectrode array is carbon.
 11. A method according toclaim 1, wherein one of said single-stranded nucleic acids is deriatizedwith a functionalized linker.
 12. A method according to claim 11,wherein said functionalized linker is comprised of 5 to 20 σ bonds. 13.A method according to claim 11, wherein said functionalized linker isthiol-terminated.
 14. A method according to claim 11, wherein saidfunctionalized linker is amine-terminated.
 15. A method according toclaim 1, wherein said addressable multielectrode array is comprised of amonolayer of oligonucleotide duplexes of 5 to 10 base-pairs in lengthdeposited onto said array, wherein each of said oligonucleotide duplexesis derivatized on one end with a functionalized linker and on theopposite end with a first single-stranded overhang of known sequencecomposition, and wherein one of said single-stranded nucleic acidscontains a second single-stranded overhang complementary to said firstsingle-stranded overhang on said electrode or addressable multielectrodearray.
 16. A method of detecting one or more base-stacking perturbationsin a target sequence comprising: (a) hybridizing a first single strandednucleic acid to a second single stranded nucleic acid to form a firstcomplex, wherein said nucleic acids are comprised of 12 to 25nucleotides, and wherein one of said single-stranded nucleic acids isderivatized with a thiol-terminated linker comprised of 5 to 20 σ bonds;(b) depositing said first complex onto an addressable goldmultielectrode array; (c) adding daumomycin to said electrode-boundfirst complex to form a second complex; and (d) measuring an electrontransfer event between said addressable gold multielectrode array anddaunomycin as an indication for the presence or absence of saidbase-stacking perturbations.
 17. A method according to claim 16, whereinsaid base-stacking perturbations are point mutations, protein-DNAadducts, adducts between any chemical entity and said target sequence,or combinations thereof.
 18. A method of detecting one or morebase-stacking perturbations in a target sequence comprising: (a)hybridizing a first single stranded nucleic acid to a second singlestranded nucleic acid to form a first complex, wherein said nucleicacids are comprised of 12 to 25 nucleotides, and wherein one of saidsingle-stranded nucleic acids is derivatized with a amine-terminatedlinker comprised of 5 to 20 σ bonds; (b) depositing said first complexonto an addressable carbon multielectrode array; (c) adding daunomycinto said electrode-bound first complex to form a second complex; and (d)measuring an electron transfer event between said addressable carbonmultielectrode array and daumonycin as an indication for the presence orabsence of said base-stacking perturbations.
 19. A method according toclaim 18, wherein said base-stacking perturbations are point mutations,protein-DNA adducts, adducts between any chemical entity and said targetsequence, or combinations thereof.
 20. A method of detecting one or morebase-stacking perturbations in a target sequence comprising: (a)hybridizing a first single-stranded nucleic acid to a secondsingle-stranded nucleic acid to form a first complex of 12 to 25nucleotides in length, wherein said first complex contains a firstsingle-stranded overhang of known sequence composition, and wherein saidfirst single-stranded overhang can be the same or different; (b)depositing said first complex onto an addressable multielectrode array,wherein said addressable multielectrode array is comprised of amonolayer of oligonucleotide duplexes of 5 to 10 base-pairs in lengthdeposited onto said array, wherein each of said oligonucleotide duplexesis derivatized on one end with a functionalized linker and on theopposite end with a second single-stranded overhang complementary tosaid first single-stranded overhang; (c) adding daumomycin to saidelectrode-bound first complex to form a second complex; and (d)measuring an electron transfer event between said addressablemultielectrode array and daunomycin as an indication for the presence orabsence of said base-stacking perturbations.
 21. A method according toclaim 20, wherein said base-stacking perturbations are point mutations,protein-DNA adducts, adducts between any chemical entity and said targetsequence, or combinations thereof.
 22. A method of detecting one or morebase-stacking perturbations electrocatalytically in a target sequencecomprising: (a) hybridizing a first single stranded nucleic acid to asecond single stranded nucleic acid to form a first complex; (b)depositing said first complex onto an electrode or an addressablemultielectrode array to form a second complex; (c) immersing said secondcomplex in a solution comprising an intercalative, redox-active speciesand a non-intercalative, redox-active species; and (d) measuring anelectron transfer event as an indication for the presence or absence ofsaid base-stacking perturbations.
 23. A method according to claim 22,wherein said base-stacking perturbations are point mutations,protein-DNA adducts, adducts between any chemical entity and said targetsequence, or combinations thereof.
 24. A method according to claim 22,wherein said intercalative, redox-active moiety is an intercalator. 25.A method according to claim 22, wherein said intercalative, redox-activemoiety is an intercalator selected from the group consisting ofphenanthridines, phenothiazines, phenazines, acridines, andanthraquinones.
 26. A method according to claim 22, wherein saidnon-intercalative, redox-active moiety is selected from the groupconsisting of ferricyanide, ferrocenes, hexacyanoruthenate, andhexacyanoosmate.
 27. A method according to claim 22, wherein saidintercalative, redox-active moiety is a protein.
 28. A method accordingto claim 22, wherein said intercalative, redox-active moiety ismethylene blue, and wherein said non-intercalative, redox-active moietyis ferricyanide.
 29. A method according to claim 22, wherein saidelectrode or addressable multielectrode array is gold.
 30. A methodaccording to claim 22, wherein said electrode or addressablemultielectrode array is carbon.
 31. A method according to claim 22,wherein one of said single-stranded nucleic acids is derivatized with afunctionalized linker.
 32. A method according to claim 31, wherein saidfunctionalized linker is comprised of 5 to 20 σ bonds.
 33. A methodaccording to claim 31, wherein said functionalized linker isthiol-terminated.
 34. A method according to claim 31, wherein saidfunctionalized linker is amine-terminated.
 35. A method according toclaim 22, wherein said addressable multielectrode array is comprised ofa monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in lengthdeposited onto said array, wherein each of said oligonucleotide duplexesare derivatized on one end with a functionalized linker and on theopposite end with a first single-stranded overhang of distinct sequencecomposition, and wherein one of said single-stranded nucleic acidscontains a second single-stranded overhang complementary to said firstsingle-stranded overhang on said electrode or addressable multielectrodearray.
 36. A method of detecting one or more point mutationselectrocatalytically within the p53 gene, comprising: (a) forming a setof oligonucleotide duplexes of approximately 20 base-pairs in lengthcorresponding to the approximately 600 base pair long region withinexons 5 through 8 of the p53 gene, wherein said oligonucleotide duplexesare derivatized with a thiol-terminated linker comprised of 5 to 20 σbonds; (b) depositing said oligonucleotide duplexes onto an addressablegold multielectrode array; (c) denaturing said oligonucleotide duplexesby immersing them in aqueous solution at elevated temperatures for 1minute and removing the complementary strands to form a single-strandedmonolayer; (d) exposing said single-stranded monolayer to a firstsamnple comprising PCR-amplified and fragmented p53 gene DNA underhybridizing conditions to form a first complex; (e) rinsing saidelectrode-bound first complex to remove any unhybridized material; (f)immersing said electrode-bound first complex into a dilute solutioncomprised of 1.0 μM methylene blue and 1.0 mM ferricyanide; (g)measuring an electron transfer event as an indication for the presenceor absence of said point mutations; (h) denaturing said electrode-boundfirst complex by immersing it in an aqueous solution at elevatedtemperatures for 1 minute, and regenerating said single-strandedmonolayer; (i) exposing said single-stranded monolayer to a secondsample containing PCR-amplified and fragmented p53 gene DNA underhybridizing conditions to form a second complex; and (k) repeating steps(e) through (h) using several sample solutions.